Method for protecting and treating at least one muscarinic receptor from dysfunction resulting from free radical damage

ABSTRACT

Methods and compositions for enhancing cellular function through protection of a tissue components such receptors, proteins, lipids, nucleic acids, carbohydrates, hormones, vitamins, and cofactors, by administering pyrophosphate analogs or related compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional application No.60/200,843 filed May 1, 2000; provisional application No. 60/233,263filed Sep. 6, 2000; and provisional application No. 60/233,025 filedSep. 15, 2000; each entitled “Methods and Compositions for EnhancingCellular Function and Protecting Receptor.”

BACKGROUND OF THE INVENTION

Cellular function depends on the maintenance of intact cellularcomponents including: receptors, proteins, lipids, nucleic acids,carbohydrates, hormones and cofactors. Cellular receptors, includingcell surface receptors, mediate communication within and between cells,tissues and organs within a living system. Cellular receptors alsoprovide a means to signal a living system, tissues, organs, cells, andsubcellular compartments. Receptors are molecules or macromolecules thatbind or interact with agents to alter or enhance their function. Manyreceptors are membrane bound proteins, which require not only that theirprotein structure be intact but also that the membrane lipids andcarbohydrates be intact and functional. Through various signalingmechanisms, the messages sent by the receptor, either in the presence orabsence of an interacting or bound agent, can be transmitted. Followingreceptor activation, signalling also requires intact cellular proteins,lipids, nucleic acids and carbohydrates in order for the message to beproperly received.

Often as a result of damage, the ability of cellular receptors tointeract with or bind various agents is decreased, resulting in animpairment of vital intrinsic and extrinsic communication. Damage tocellular receptors and other cellular components diminishes the abilityof a receptor to bind agents and elicit a communication or signalingevent. This can result in damage or death to cells, resulting in damageor diseases of tissues, organs and living systems. Accordingly, there isa need for a means to protect receptors and other cellular componentsfrom damage and to increase the efficacy of agents that exert theireffects through cellular receptors.

SUMMARY OF THE INVENTION

The invention provides methods for enhancing cellular function throughprotection of tissue components and/or increasing the efficacy of atherapeutic agent in a subject in need thereof. The method includesadministering a composition, such as a pharmaceutical composition, of apyrophosphate analog. In a second embodiment, the method includesadministering a composition, such as a pharmaceutical composition, of aprotective agent.

Preferably, the invention provides a method for protecting a muscarinicacetylcholine receptor (mAChR) and/or increasing the efficacy of anagent that directly or indirectly affects a mAChR in a subject in needthereof. Suitable agents that directly or indirectly affect a muscarinicreceptor include anticholinesterase agents, muscarinic agonists,allosteric regulators of a muscarinic receptor, muscarinic antagonists,and neurotrophic and neuritogenic factors that are similar to naturallyoccurring nerve growth promoting substances. In one embodiment, theinvention provides a method to protect a mAChR and/or increase theefficacy of agents that directly or indirectly affect a mAChR in thecentral nervous system (CNS) of a subject in need thereof. Preferably, amuscarinic receptor is protected from an endogenous low molecular weightinhibitor from Alzheimer's brain tissue, a metal, or oxidative stress.In another embodiment, the invention provides a method to protect amAChR and/or increase the efficacy of agents that directly or indirectlyaffect a mAChR not in the CNS of a subject in a need thereof. In a firstembodiment, the method includes administering a pyrophosphate analog. Ina second embodiment, the method includes administering a protectiveagent.

The invention also provides a method for increasing the efficacy of atherapeutic agent, preferably a neurologic agent, in a subject in needthereof. In a first embodiment, this method includes administering apyrophosphate analog. In this embodiment, the increased efficacy of theneurologic agent preferably results from protection of a muscarinicreceptor caused or induced by the pyrophosphate analog. In a secondembodiment, this method includes administering a protective agent. Inthis second embodiment, the increased efficacy of the neurologic agentpreferably results from protection of a muscarinic receptor caused orinduced by the protective agent. In each embodiment, the subjectpreferably is concurrently receiving, has recently received, or willsoon receive a neurologic agent such as nerve growth factor (NGF),insulin growth factor (IGF-1), brain derived neurotrophic factor (BDNF),fibroblast growth factor (FGF), and the like; certain other knownneurotrophins and neuroprotectants; and medications used for stroke,Alzheimer's disease, Parkinson's disease, ALS, traumatic brain or spinalcord injury, cancer, diabetes, neuropathies, hypertension, irritablebowel syndrome; diseases or disorders of the heart and smooth muscles,blood, blood vessels, glands or bones and other disorders. Preferably,the therapeutic agent directly or indirectly affects a mAChR. Suchagents include an anticholinesterase agent, a muscarinic agonist, and amuscarinic antagonist.

Pyrophosphate analogs that can be employed in the appropriate embodimentof the method of the invention include compounds of Formula I:

where each X is independently O, CH₂, NH, or S; R¹ is H, a small alkylgroup, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,serine, threonine, tyrosine, arachidonyl, —PO(OH)(OR²), or—(PO(OH)O)_(m)—PO(OH)(OR²), and m is 1-3; R² is H, a small alkyl group,guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol, serine,threonine, tyrosine, or arachidonyl; and n is 1-900. Compounds ofFormula I in which R¹ is a small alkyl group, guanyl, adenylyl,glycerol, acyl glycerol, diacyl glycerol, serine, threonine,arachidonyl, —PO(OH)(OR²), or —(PO(OH)O)_(m)—PO(OH)(OR²); or R² is asmall alkyl group, guanyl, adenylyl, glycerol, acyl glycerol, diacylglycerol, serine, threonine, tyrosine, or arachidonyl can be referred toas substituted pyrophosphate analogs. Compounds of Formula I can alsoinclude substituted pyrophosphate analogs such as adinucleoside-5-5′-pyrophosphate, a cyclopyrophosphate of purine, and apyrimidine acyclonucleoside. The compound of Formula I can be anypharmaceutically acceptable salt or basic addition salt. Preferably, Xis O, CH₂, NH, or S; R¹ is H; R² is H; and n is 1-6. More preferably thepyrophosphate analog is pyrophosphate or imidodiphosphate.

Additional pyrophosphate analogs include compounds of Formula II:

where n=2-4; X is O, RCR¹; CR; C (n=4), CH (n=3), or CH₂ (n=2); NH; N;S; and R and/or R¹ is H, OH, a small alkyl group, such as CH or(CH₂)_(m)NH₂ where m=1-6. Further included are bisphosphonic acids,which are also known as bisphosphonates, where X is preferably RCR¹,where R and R¹ groups are chosen independently from OH, H₂N(CH₂)₂, orCH₃. For example, RCR¹ can be H₂N(CH₂)₂C(OH) or CH₃COH. Morespecifically, the bisphosphonates include etidronic acid((1-Hydroxyethylidene)bisphosphonic acid) and pamidronic acid((3-Amino-1-hydroxypropylidene)bisphosphonic acid) where preferably n=2.

Yet more additional pyrophosphate analogs include substitutedpyrophosphate analogs such as an inositol diphosphate, an inositoltriphosphate, an inositol tetraphosphate, an inositol pentaphosphate,and an inositol hexaphosphate.

Suitable protective agents that can be employed in an embodiment of themethod of the invention include a bilirubin, biliverdin, carnosol,quercetin, myricetin, a bioflavinoid, a combination thereof, or apharmaceutically acceptable salt thereof; a heme binding compound, suchas hemopexin, lipopexin, a lipoprotein, or ApoE-2; and a heme oxygenase,such as heme oxygenase-1 or heme oxygenase-2, biliverdin reductase, acatalase, a peroxidase, a vector encoding a biliverdin reductase, avector encoding a heme oxygenase (e.g. a vector encoding a hemeoxygenase-1 or a vector encoding a heme oxygenase-2), a vector encodinga catalase, a vector encoding a peroxidase, or a combination thereof.Biliverdin reductase can be administered alone or in combination with aheme oxygenase. Heme oxygenases include recombinant heme oxygenase.Preferably, a heme oxygenase is a human heme oxygenase.

The method of the invention can treat or prevent a CNS disorder.Preferably, the method of the invention can treat or preventneurodegeneration, can improve memory and cognition, can treat orprevent brain deterioration or cognitive and memory loss associated withaging, or can treat or prevent Alzheimer's Disease, Parkinson's disease,Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, affective disorders,anxiety disorders, and/or schizophrenia; nerve damage fromcerebrovascular disorders such as stroke or atherosclerosis in the brainor spinal cord, from CNS infections including meningitis and HIV, fromtumors of the brain and spinal cord, prion diseases, and CNS disordersresulting from ordinary aging (e.g., anosmia), brain injury, or spinalcord injury.

In another embodiment, the method of the invention can treat or preventa disease or disorder not of the CNS. Preferably, the method of theinvention can treat or prevent cancer, or neuropathies or diseases ordisorders of the heart, smooth muscles, blood, blood vessels, glands, orbones. Such diseases or disorders include hypertension, myocardialinfarction, ischemic heart disease, congestive heart failure, cardiacarrhythmias, cancer, irritable bowel syndrome, diverticular disease,urinary incontinence, esophageal achalasia, chronic obstructive airwaysdisease, xerostomia, diabetes mellitus, Sjogren's syndrome or dry eyesyndrome which involves decreased secretion of tears by, for example,the lacrimal glands, Paget's disease, hereditary hematochromatosis or anon-CNS disorder resulting from normal aging.

In another embodiment, the method of the invention treats infections,including (without limitation) bacterial, fungal, algo, or algaeinfections. Such infections can occur in plants (for which a preferredembodiment of the invention employs imidodiphosphate as thepyrophosphate analog), animals, or mammals.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates protection of a mAChR by pyrophosphate. Pyrophosphateprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Pyrophosphate protected the receptor from loss ofantagonist (³H-QNB (quinulidinyl benzilate)) binding.

FIG. 2 illustrates protection of a mAChR by pyrophosphate. Pyrophosphateprotected the mAChR from inactivation by heme and peroxide.Pyrophosphate protected the receptor from loss of antagonist (³H-QNB)binding.

FIG. 3 illustrates protection of a mAChR by pyrophosphate. Pyrophosphateprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Pyrophosphate protected the receptor from loss ofagonist (oxotremorine) binding.

FIG. 4 illustrates protection of a mAChR by imidodiphosphate.Imidodiphosphate protected the mAChR from inactivation by the endogenouslow molecular weight inhibitor. Imidodiphosphate protected the receptorfrom loss of antagonist (³H-QNB) binding.

FIG. 5 illustrates protection of a mAChR by guanylimidodiphosphate.Guanylimidodiphosphate protected the mAChR from inactivation by theendogenous low molecular weight inhibitor. Guanylimidodiphosphateprotected the receptor from loss of antagonist (³H-QNB) binding.

FIG. 6 illustrates protection of a mAChR by adenylylimidodiphosphate.Adenylylimidodiphosphate protected the mAChR from inactivation by theendogenous low molecular weight inhibitor. Adenylylimidodiphosphateprotected the receptor from loss of antagonist (³H-QNB) binding.

FIG. 7 illustrates protection of a mAChR by tripolyphosphate.Tripolyphosphate protected the mAChR from inactivation by the endogenouslow molecular weight inhibitor. Tripolyphosphate protected the receptorfrom loss of antagonist (³H-QNB) binding.

FIG. 8 illustrates protection of a mAChR by bilirubin. Bilirubinprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Bilirubin protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 9 illustrates protection of a mAChR by bilirubin. Bilirubinprotected the mAChR from inactivation by heme and peroxide. Bilirubinprotected the receptor from loss of antagonist (³H-QNB) binding.

FIG. 10 illustrates protection of a mAChR by bilirubin. Bilirubinprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Bilirubin protected the receptor from loss of agonist(oxotremorine) binding.

FIG. 11 illustrates protection of a mAChR by biliverdin. Biliverdinprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Biliverdin protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 12 illustrates protection of a mAChR by carnosol, Carnosolprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Carnosol protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 13 illustrates protection of a mAChR by quercetin. Quercetinprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Quercetin protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 14 illustrates protection of a mAChR by myricetin. Myricetinprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Myricetin protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 15 illustrates protection of a mAChR by catalase. Catalaseprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor. Catalase protected the receptor from loss ofantagonist (³H-QNB) binding.

FIG. 16 illustrates protection of a mAChR by catalase. Catalaseprotected the mAChR from inactivation by heme and peroxide. Catalaseprotected the receptor from loss of antagonist (³H-QNB) binding.

FIG. 17 illustrates protection of a mAChR by a peroxidase. Theperoxidase protected the mAChR from inactivation by the endogenous lowmolecular weight inhibitor. The peroxidase protected the receptor fromloss of antagonist (³H-QNB) binding.

FIG. 18 illustrates protection of a mAChR by a peroxidase. Theperoxidase protected the mAChR from inactivation by heme and peroxide.The peroxidase protected the receptor from loss of antagonist (³H-QNB)binding.

FIG. 19 illustrates protection of mAChR by pamidronate. Pamidronateprotected the mAChR from inactivation by the endogenous low molecularweight inhibitor.

FIG. 20 illustrates protection of a mAChR by pyrophosphate.Pyrophosphate protected the mAChR from damage by the metal lead in theform of PbCl₂. Pyrophosphate protected the receptor from loss ofantagonist (³H-QNB (quinulidinyl benzilate)) binding.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “cholinesterase” refers to an enzyme capable ofhydrolyzing acetylcholine and includes acetylcholinesterase.

As used herein, “agonist” refers to an agent that binds to or interactswith a receptor and elicits a response transduced through the receptor.Agonist includes full agonists, partial agonists, and inverse agonists.A full agonist is an agent that can elicit a maximal response from areceptor. A partial agonist is an agent that can elicit, at best, a lessthan maximal response from a receptor. An inverse agonist is an agentthat produces a response that is opposite that of a full or partialagonist. For example, if agonist binding or interaction with a receptorresults in increased concentration of cAMP within a cell, then inverseagonist binding or interaction with the same receptor will result in adecreased concentration of cAMP within the cell.

As used herein, “antagonist” refers to an agent that is capable ofpartially or completely inhibiting, or reversing, the effect of anagonist on a receptor.

As used herein, “allosteric modifier” refers to an agent that binds orinteracts with a site other than the agonist binding site of a receptorand modifies the ability of an agonist or an antagonist to elicit orinhibit, respectively, a response transduced through a receptor, withoutitself eliciting a response.

As used herein, “tissue component” includes receptors, proteins, lipids,nucleic acids, carbohydrates, hormones, vitamins, and cofactors.

As used herein, “receptor” refers to any molecule or macromoleculewithin or on a cell that interacts with another molecule ormacromolecule to confer a response or transduce a signal and includesnuclear receptors, mitochondrial receptors, cytoplasmic receptors, andcell surface receptors. Receptors include receptors for neurotrophins(including, without limitation, nerve growth factor, neurotrophins 3, 4,and/or 5 (NT-3, NT-4 and/or NT-5) and brain derived growth factors);neurotransmitters; hormones; steroids; local mediators such as nitricoxide, carbon monoxide, histamine, and growth factors like insulin,insulin-like growth factor-I, fibroblast growth factors, cilliaryneurotrophic factor, glia-derived neurotrophic factor, glia-derivednexin, cholinergic enhancing factor, transforming growth factors,activity dependent neurotrophic factor, neurokines, gangliosides,phosphatidylserine, PDGF (platelet derived growth factor) and EGF(epidermal growth factor); benzodiazepines; arachidonic acid; purines(including, without limitation, adenosine and ATP); nucleotides andcyclic nucleotides; calcium and other divalent cations; odorants;antisense oligonucleotides; opiates; cannabinoids; glutamate; melatonin;angiotensin II; secretin; vasoactive intestinal peptide;cholecystokinin; ACTH; vasopressin; thrombin; ion channels; and thelike. Receptors also include but are not limited to G-protein-coupledreceptors, ion-channel-linked receptors and enzyme-linked receptors.

As used herein, “protecting a receptor” refers to protecting thephysical integrity of a receptor and/or the function of a receptor, suchas enhancing the function of a receptor; or maintaining the ability ofthe receptor to respond to agonists, to respond to antagonists, totransmit a message to the interior of a cell, or to send a signal withina cell, cell nucleus, or mitochondria.

As used herein, “central nervous system” (CNS) refers to the brain andspinal cord and associated tissues.

As used herein, “disorders and diseases of the CNS” refers to braindiseases such as Alzheimer's disease, Parkinson's disease, Lewy bodydementia, multiple sclerosis, cerebellar ataxia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, affective disorders,anxiety disorders, and/or schizophrenia; cell damage; nerve damage fromcerebrovascular disorders such as stroke in the brain or spinal cord,from CNS infections including meningitis and HIV, from tumors of thebrain and spinal cord, prion diseases, and CNS disorders resulting fromordinary aging (e.g., anosmia), brain injury, or spinal cord injury.

As used herein, a disease or disorder that relates to or is caused atleast in part by dysfunction, alteration, or loss of one or moreG-protein coupled receptors refers to Alzheimer's disease; Parkinson'sdisease; drug addiction, such as opiate addiction or cannabinoid abuse;pain; Sjogren's or dry eye syndrome; heart diseases including congestiveheart failure, myocardial infarction, cardiac arrhythmia; diseases ofsmooth muscle organs or glands such as irritable bowel syndrome,colitis, hypertension, erectile dysfunction, diabetes, obesity, bloodcoagulation disorders; and the like.

An “effective amount” of agent is an amount sufficient to prevent,treat, reduce and/or ameliorate the symptoms and/or underlying causes ofany of the above disorders or diseases. In some instances, an “effectiveamount” is sufficient to eliminate the symptoms of those diseases and,perhaps, overcome the disease itself. Preferably, an effective amount ofan agent yields a tissue concentration in the range of about 10⁻⁷ molarto about 10⁻⁵ molar, but the concentrations may be greater provided thattoxicity is avoided.

In the context of the present invention, the terms “treat” and “therapy”and the like refer to alleviate, slow the progression, prophylaxis,attenuation or cure of existing disease. Prevent, as used herein, refersto putting off, delaying, slowing, inhibiting, or otherwise stopping,reducing or ameliorating the onset of such diseases or disorders. It ispreferred that a large enough quantity of the agent be applied innon-toxic levels in order to provide an effective level of activityagainst the disease. The method of the present invention may be usedwith any animal, such as a mammal or a bird (avian), more preferably amammal. Poultry are a preferred bird. Exemplary mammals include, but arenot limited to rats, cats, dogs, horses, cows, sheep, pigs, and morepreferably humans.

Protecting a Tissue Component

The invention provides a method for protecting any biomolecule or tissuecomponent including a protein, a lipid, a nucleic acid, a carbohydrate,a hormone and the like. The invention is best illustrated, but is notlimited to, the example of the protection of a receptor, preferably amuscarinic receptor, preferably a mAChR. In a first embodiment, themethod includes administering a pyrophosphate analog. In a secondembodiment, the method includes administering a protective agent.Protecting a receptor includes protecting the physical integrity of areceptor and/or the function of a receptor, such as maintaining theability of the receptor to respond to agonists, to respond toantagonists, to transmit a message to the interior of a cell, or to senda signal within a cell, cell nucleus, or mitochondria.

An embodiment of the invention provides a method for protecting areceptor from free radical damage. Free radicals and other reactiveoxygen species (e.g., H₂O₂, HOCl, and radicals such as O₂ ⁻, sulfurcation, nitric oxide radical, ferryl, peroxyl, peroxynitrite, thiyl,thiylperoxyl, and alkoxyl) are highly reactive, and many free radicalreactions are highly damaging to cellular components. Free radicalreactions can crosslink proteins, mutagenize DNA, and peroxidize lipids.Such reactions can have deleterious effects on cellular receptors.Preferably, the method of the invention includes protection of areceptor, such as a mAChR, or of DNA, RNA, lipids, and proteinsnecessary for receptor function from deleterious effects.

In another embodiment, the invention provides a method for reducing oreliminating deleterious effects of an endogenous inhibitor found inelevated levels in the brains of Alzheimer's disease patients. Thisendogenous, low molecular weight inhibitor, as it is known, inhibitsagonist and antagonist binding to mAChRs. This inhibitor has a molecularweight of less than 3500 Da and is believed to generate free radicals,in the presence of glutathione or other sulthydryl compounds, thatirreversibly inhibit or inactivate the mAChR The inhibitor also containsfree heme, which can generate free radicals, including superoxideradicals, peroxyl radicals, and thiyl radicals, and can causeneurotoxicity. Heme has been shown to damage protein and lipidcomponents of membranes by Vincent (Oxidative Effects of Heme andPorphyrins on Proteins and Lipids, Seminars in Hematology 26(2):105-113, 1989). Membrane lipid defects have been demonstrated inAlzheimer's disease by Ginsberg et al. (Evidence for a Membrane LipidDefect in Alzheimer's Disease, Mol. and Chem. Neuropathol. 19: 37-46,1993). In addition, heme has been proposed to contribute toatherosclerosis by Jacob (Newly recognized causes of atherosclerosis:The role of microorganisms and of vascular iron overload, J. Lab. Clin.Med. 123: 808-816, 1994).

In one embodiment, the method of the invention includes increasing theefficacy of an agent that directly or indirectly affects a mAChR. By wayof example, administration of a pyrophosphate analog can increase theefficacy of a muscarinic agonist in the presence of the inhibitor. Inanother embodiment, the method of the invention includes reducing oreliminating deleterious effects the low molecular weight inhibitor orheme by decreasing or preventing the generation of free radicals ortrapping radicals once formed.

Receptors

The invention provides a method for protecting a receptor and/orincreasing the efficacy of agents that directly or indirectly affect areceptor. Such receptors include G-protein-coupled receptors,ion-channel-linked receptors and enzyme-linked receptors. Examplesinclude receptors for neurotrophins; neurotransmitters; hormones;steroids; local mediators such as nitric oxide, histamine, and growthfactors like PDGF (platelet derived growth factor) and EGF (epithelialgrowth factor); nucleotides and cyclic nucleotides; calcium and otherdivalent cations; odorants; antisense oligonucleotides; and the like.Preferably the receptor is a muscarinic receptor. Examples of G-proteincoupled receptors include receptors that respond to odorants, opiates,cannabinoids, glutamate, melatonin, angiotensin II, secretin, vasoactiveintestinal peptide (VIP), cholecystokinin (CCK), adrenaline (adrenergicreceptors), acetylcholine (muscarinic receptors), ACTH, vasopressin,thrombin, and the like.

In one embodiment the invention provides a method for protecting areceptor and/or increasing the efficacy of agents that directly orindirectly affect a receptor in the CNS. In another embodiment, theinvention provides a method for protecting a receptor and/or increasingthe efficacy of agents that directly or indirectly affect a receptor notin the CNS. Agents whose efficacy are increased by the method of theinvention include receptor agonists, allosteric modifiers of receptors,and receptor antagonists.

An embodiment of the invention provides a method for treating orpreventing a disease or disorder that relates to or is caused at leastin part by dysfunction, alteration, or loss of one or more G-proteincoupled receptors. These diseases and disorders include Alzheimer'sdisease; Parkinson's disease; stroke; multiple sclerosis; ALS; drugaddiction, such as opiate addiction or cannabinoid abuse; pain;Sjogren's or dry eye syndrome; heart diseases including congestive heartfailure, myocardial infarction, cardiac arrhythmia; cancer; diseases ofsmooth muscle organs or glands such as irritable bowel syndrome,colitis, hypertension, erectile dysfunction, diabetes, obesity, bloodcoagulation disorders; and the like.

Muscarinic Receptors

The invention provides a method for protecting a mAChR and/or increasingthe efficacy of agents that directly or indirectly affect a mAChR. Thereare at least five pharmacological classes of mAChRs, including the M1,M2, and M3 muscarinic receptors, and several genetic subclassesincluding m1, m2, m3, m4, and m5. These muscarinic receptors areG-protein coupled receptors. Each receptor subtype has its own uniquepattern of expression throughout various tissues. As such, dysfunctionof each receptor subclass, or combinations thereof, may have deleteriouseffects leading to a variety of diseases or disorders. The method of theinvention can provide protection to a muscarinic receptor in any orseveral mAChR subclasses, and therefore, can be of benefit to those atrisk or suffering from diseases associated with dysfunction of one ormore muscarinic receptor subtype. Preferably, the method of theinvention provides protection to M1 and M2 muscarinic receptors.

Muscarinic receptors mediate numerous of the inhibitory and excitatoryeffects of the neurotransmitter acetylcholine in the heart, smoothmuscle, blood vessels, glands and in neurons (both presynaptic andpostsynaptic) in the autonomic and the central nervous system.Dysfunction of mAChRs thus can contribute to a variety of diseasesand/or disorders. Through protection of a mAChR and/or throughincreasing the efficacy of agents that directly or indirectly affect amAChR, the method of the invention can provide benefit to subjectssuffering from or at risk of a disease or disorder associated with mAChRdysfunction.

An embodiment of the invention provides a method for protecting a mAChRand/or increasing the efficacy of agents that directly or indirectlyaffect a mAChR in the nervous system of a subject, and therefore, can beof benefit to subjects suffering from or at risk of central nervoussystem or peripheral nervous system disorders. For example, mAChRs andother receptors are involved in the regulation of the function of cellsthroughout the CNS. Accordingly, the method of the invention can providebenefit to subjects suffering from or at risk of CNS disorders such asAlzheimer's disease, Parkinson's disease, Lewy body dementia, multiplesclerosis, cerebellar ataxia, progressive supranuclear palsy,amyotrophic lateral sclerosis, affective disorders, anxiety disorders,and/or schizophrenia; nerve damage from cerebrovascular disorders suchas stroke, from CNS infections including meningitis and HIV, from tumorsof the brain and spinal cord, prion diseases, and CNS disordersresulting from ordinary aging, brain injury, or spinal cord injury.mAChRs are also involved in the regulation of the function of cells inthe peripheral nervous system, including the autonomic nervous system.Accordingly, the method of the invention can provide benefit to subjectssuffering from or at risk of peripheral nervous system disorders, suchas peripheral neuropathy, including that associated with diabetes. Forexample, in diabetic patients nerves can deteriorate as blood vesselsthat contain muscarinic or other receptors are lost. Preferably, themethod of the invention can benefit a subject suffering from or at riskof Alzheimer's disease.

Another embodiment of the invention provides a method for protecting amAChR and/or increasing the efficacy of agents that directly orindirectly affect a mAChR not within the nervous system of a subject,and therefore, can be of benefit to subjects suffering from or at riskof disease or disorder outside the nervous system. For example, mAChRsare involved in the regulation (e.g., stimulation or inhibition) ofsmooth muscle contraction, the regulation of heart rate and cardiaccontractility, the regulation of secretion of enzymes or hormones,including the release of amylase from the parotid gland and the releaseof digestive enzymes and insulin from the pancreas, the regulation ofbone growth, and the regulation of iron metabolism. Accordingly, themethod of the invention can provide benefit to subjects suffering fromor at risk of smooth muscle related disorders such as irritable bowelsyndrome, diverticular disease, urinary incontinence, esophagealachalasia, diseases or disorders of the blood vessels (e.g.hypertension), or chronic obstructive airways disease; heart musclerelated disorders such as pathologic bradycardia or tachycardia,arrhythmia, flutter or fibrillation; blood related disorders such ashereditary hematochromatosis; bone disorders such as Paget's disease;cancer; and gland related disorders such as xerostomia, diabetesmellitus, or Sjogren's syndrome or dry eye syndrome which involvesdecreased secretion of tears by, for example, the lacrimal glands. Forexample, tear secretion is known to require muscarinic cholinergicstimulation and intact muscarinic receptors. Accordingly, protection ofa mAChR and/or increasing the efficacy of an agent that directly orindirectly affects a mAChR can be of benefit to a subject suffering fromSjogren's syndrome or dry eye syndrome.

Protecting a muscarinic receptor can result in enhancing theeffectiveness of agents that directly or indirectly affect a mAChR.Useful agents that affect a mAChR include, but are not limited to,anticholinesterase agents, muscarinic agonists, muscarinic antagonists,and other agents useful for treatment of diseases associated withdysfunction of muscarinic receptors, including neurodegenerative andother CNS disorders.

The method of the invention also provides enhanced efficacy of agentsthat do not act directly, or indirectly, with a mAChR. Such enhancedefficacy can be achieved, for example, through protection of a receptor,preferably a mAChR. Protecting a muscarinic receptor can result inenhancing the efficacy of agents that do not exert their action directlyor indirectly on the muscarinic receptor. Such enhanced efficacy can beachieved through desirable effects on cells to which protection ofmuscarinic receptors provides benefit. Typically, cells that derivebenefit from protection of a muscarinic receptor are cells that containmuscarinic receptors. Examples of cells that contain a mAChR includeparticular neurons, smooth muscle cells, and gland cells.

Cells that lack a muscarinic receptor but which interact with enzymes,hormones, and/or other compounds released from cells with a muscarinicreceptor can derive benefit from protecting a muscarinic receptor.Examples of cells lacking a mAChR that can derive benefit fromprotecting a mAChR include cells that are presynaptic or postsynapticrelative to cells that contain a mAChR and cells that can interact withenzymes, hormones, and/or other compounds released from cells thatcontain a mAChR.

Examples of this phenomenon include: Stimulation of m₂ receptors onpresynaptic membranes increases the release of acetylcholine which canthen stimulate nicotinic receptors on other post synaptic cells.Stimulation of mAChR releases arachidonic acid, which can then affect avariety of other nearby brain cells. Arachidonic acid also increasessecretion of amyloid precursor protein. Emmerling, M. R. et al. (1996)Ann. N.Y. Acad. Sci. 777:310-315. Activation of m1 and m3 mAChRattenuates release of amyloid B protein. Hung, A. Y. et al. (1993) J.Biol. Chem. 268:22959-22962. Stimulation of mAChR is required for memoryand learning which also involve the proper function of noncholinergiccells. Stimulation of mAChR can increase the nitric oxide-cyclic GMPsignaling system in neurons (Bauer, M. B. (1994) Neuroscience62:351-359) and nitric oxide can travel from one cell to another toproduce its effects. Stimulation of mAChRs markedly increase hippocampalBDNF and NGF in RNA levels. Once produced, these neurotrophins canproduce important effects on other nerve cells in the brain. M. da PenhaBerzaghi (1993) J. Neuroscience 13(9) 3818-3826.

By way of further example, some cells containing a mAChR in the pancreascan release insulin. Released insulin can then interact with cells inclose proximity to, or at relatively great distances from, the cell fromwhich it was released. Protection of a mAChR on a cell in the pancreasthat releases insulin can have beneficial effects on a cell thatinteracts with insulin. Thus, cells lacking a mAChR can benefit fromprotection of a mAChR. Similarly, the efficacy of agents acting on cellslacking a mAChR can be enhanced by the method of the invention.

By way of yet further example, stimulation of mAChR in certain braincells increases potassium ion evoked release of the neurotransmitterdopamine which then goes on to affect other brain cells having dopaminereceptors. Joseph, J. A. et al. (1995) Brain Res. 673:195-193. Thus, CNScells lacking a mAChR can benefit from protection of a mAChR. Similarly,the efficacy of agents acting on CNS cells lacking a mAChR can beenhanced by the method of the invention.

Pyrophosphate Analogs

In one embodiment, the method of the invention provides protection to areceptor and/or increases the efficacy of agents by administering to asubject a pyrophosphate analog. Useful pyrophosphate analogs includecompounds of Formula I:

where each X is independently O, CH₂, NH, or S; R¹ is H, a small alkylgroup, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,serine, threonine, tyrosine, arachidonyl, —PO(OH)(OR²), or—(PO(OH)O)_(m)—PO(OH)(OR²), and m is 1-3; R² is H, a small alkyl group,guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol, serine,threonine, tyrosine, or arachidonyl; and n is 1-900. Compounds ofFormula I in which R¹ is a small alkyl group, guanyl, adenylyl,glycerol, acyl glycerol, diacyl glycerol, serine, threonine,arachidonyl, —PO(OH)(OR²), or —(PO(OH)O)_(m)—PO(OH)(OR²); or R² is H,guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol, serine,threonine, tyrosine, or arachidonyl can be referred to as substitutedpyrophosphate analogs. Compounds of Formula I can also includesubstituted pyrophosphate analogs such asdinucleoside-5-5′-pyrophosphates, cyclophosphates of purine andpyrimidine acyclonucleosides. The compound of Formula I can be anypharmaceutically acceptable salt or basic addition salt. Preferably, Xis O, CH₂, NH, or S; R¹ is H; and n is 2-6. More preferably thepyrophosphate analog is pyrophosphate or imidodiphosphate.

Additional preferred compounds of Formula I include pyrophosphate,glycerol pyrophosphate, arachidonylpyrophosphate, imidodiphosphate,serine phosphate, serine imidophosphate, threonine phosphate, threonineimidophosphate, guanylimidodiphosphate and adenylylimidodiphosphate.More preferably compounds of Formula I include pyrophosphate,imidodiphosphate, guanylimidodiphosphate and adenylylimidodiphosphate.

Additional pyrophosphate analogs include compounds of formula II:

where n=2-4; X is O; RCR¹; CR; C (n=4), CH (n=3), or CH₂ (n=3); NH; N;S; and R and/or R¹ is H, OH, a small alkyl group (such as CH₃), or(CH₂)_(m)NH₂ where m=1-6. Further included are bisphosphonic acids,which are also known as bisphosphonates, where X is preferably RCR¹ andR and R¹ groups are chosen independently from OH, H₂N(CH₂)₂, or CH₃. Forexample, RCR¹ can be H₂N(CH₂)₂C(OH) or CH₃COH. More specifically, thebisphosphonates include etidronic acid((1-Hydroxyethylidene)bisphosphonic acid) and pamidronic acid((3-Amino-1-hydroxypropylidene)bisphosphonic acid) where preferably n=2.

Yet more additional pyrophosphate analogs include substitutedpyrophosphate analogs such as inositol diphosphate, inositoltriphosphate, inositol tetraphosphate, inositol pentaphosphate, andinositol hexaphosphate.

Such pyrophosphate and imidodiphosphate compounds and the like can beprepared as basic addition salts, such as sodium, potassium, ormagnesium salts. It is believed that the use of a basic addition salt,such as a magnesium salt, will reduce the charge and allow for freermovement of the compound throughout the body. Pyrophosphate compounds,imidopyrophosphate compounds, and the like can be covalently bound toother phosphates creating polyphosphates or polyimidophosphates. One ormore pyrophosphate analogs can be administered in combination. Inanother embodiment, the pyrophosphate analog can be administered with aprotective agent. In another embodiment, the pyrophosphate analog can beadministered with a neurologic agent, and optionally with a protectiveagent.

Protective Agents

In another embodiment, the invention provides a method for protectingreceptors and/or increasing the efficacy of agents by administering to asubject a protective agent. Protective agents useful in an embodiment ofthe method of the invention include a bilirubin, biliverdin, carnosol,quercetin, myricetin, a bioflavinoid; a heme binding compound, such ashemopexin, lipopexin, a lipoprotein, or ApoE-2; and a heme oxygenase,such as heme oxygenase-1 or heme oxygenase-2, or biliverdin reductase, acatalase, a peroxidase, a DNA or RNA vector encoding a biliverdinreductase, a DNA or RNA vector encoding a heme oxygenase (e.g. a DNA orRNA vector encoding a heme oxygenase-1 or a DNA or RNA vector encoding aheme oxygenase-2), a DNA or RNA vector encoding a catalase, a DNA or RNAvector encoding a peroxidase, or a combination thereof. Biliverdinreductase is preferably administered with bilirubin, because biliverdinreductase can regenerate bilirubin from biliverdin after bilirubin hasbeen oxidized while functioning as a protective agent. Biliverdinreductase is also preferably administered in combination with a hemeoxygenase. Heme oxygenases include recombinant heme oxygenase.Preferably, a heme oxygenase is a human heme oxygenase.

One or more protective agents can be administered in combination. Inanother embodiment, one or more protective agents can be administered incombination with one or more pyrophosphate analogs. In anotherembodiment, one or more protective agents can be administered in withone or more neurologic agents, and optionally with one or morepyrophosphate analogs.

Agents that Directly or Indirectly Affect a mAChR

The invention also provides a method for enhancing the efficacy of oneor more agents that directly or indirectly affect a mAChR. Agents thatdirectly or indirectly affect a mAChR include agents that (1) bind to orinteract with a mAChR to either elicit a response transduced through amAChR or reduce or prevent an agent from binding to or interacting witha mAChR and/or eliciting a signal transduced through a mAChR, (2) alterthe concentration of agents that bind to or interact with a mAChR toeither elicit a response transduced through a mAChR or reduce or preventan agent from binding to or interacting with a mAChR and/or eliciting asignal transduced through a mAChR, or (3) modify the ability of agentsthat bind to or interact with a mAChR to either elicit a responsetransduced through a mAChR or reduce or prevent an agent from binding toor interacting with a mAChR and/or eliciting a signal transduced througha mAChR. Such agents include anticholinesterase agents, muscarinicagonists, muscarinic antagonists, and allosteric modifiers of muscarinicreceptors. Preferably, the invention provides a method for enhancing theefficacy of agents that either directly or indirectly elicit a responsethrough a mAChR. Most preferably, the method of the invention enhancesthe efficacy of a muscarinic agonist or an anticholinesterase agent.

Muscarinic receptor agonists directly elicit a response through a mAChRby binding to and transducing a signal through a mAChR. Preferredmuscarinic agonists include acetylcholine, Xanomeline, and the like.

A muscarinic antagonist is an agent that is capable of partially orcompletely inhibiting, or reversing, the effect of a muscarinic agoniston a mAChR. Examples of muscarinic antagonists include atropine,N-methyl-scopolamine, quinuclidinyl benzilate, pirenzepine, and thelike.

Cholinesterase hydrolyzes the neurotransmitter acetylcholine andprovides one of the mechanisms responsible for rapid depletion ofacetylcholine from the synaptic cleft. Anticholinesterase agents inhibitcholinesterase activity and as a result increase the concentration ofacetylcholine in the synaptic cleft and prolong the duration of whichacetylcholine remains in the synaptic cleft. Anticholinesterase agentscan thus indirectly affect a mAChR by increasing concentrations of andprolonging the effective duration of acetylcholine in the synapticcleft. Anticholinesterase agents can also interact directly withcholinergic receptors, including mAChRs; with sodium and potassium ionchannels; and effect the uptake, synthesis and release ofneurotransmitters. Preferred anticholinesterase agents include Aricept,Exelon, Metrifonate, and the like.

An allosteric modifier of a mAChR binds or interacts with a site otherthan the agonist binding site of a mAChR and modifies the ability of anagonist or an antagonist to elicit or inhibit, respectively, a responsetransduced through a muscarinic receptor, without itself eliciting aresponse. Suitable allosteric modifiers of a muscarinic agonist includegallamine and dynorphin. Preferred allosteric modifiers of a mAChRinclude gallamine and dynorphin.

Neurologic Agents

The invention also provides a method for increasing the efficacy of aneurologic agent in a subject in need thereof. In a first embodiment,this method includes administering a pyrophosphate analog. In thisembodiment, the increased efficacy of the neurologic agent preferablyresults from protection of a muscarinic receptor caused or induced bythe pyrophosphate analog. In a second embodiment, this method includesadministering a protective agent. In this second embodiment, theincreased efficacy of the neurologic agent preferably results fromprotection of a muscarinic receptor caused or induced by the protectiveagent. In each embodiment, the subject preferably is concurrentlyreceiving, has recently received, or will soon receive a neurologicagent.

A neurologic agent promotes nerve cell growth and survival or augmentsthe activity of functioning cells. Among those agents that are preferredare cholinergic agonists, allosteric modifiers of a mAChR,cholinesterase inhibitors, or neurotrophic and neuritogenic factors thatare similar to naturally occurring nerve growth promoting substances.Among the preferred neurologic agents are gangliosides (such as GM-Iganglioside), phosphatidylserine (PS), nerve growth factor (NGF),neurotrophins 3, 4, and/or 5(NT-3, NT-4 and/or NT-5) brain-derivedneurotrophic factor (BDNF), fibroblast growth factors (FGFs, e.g., basicfibroblast growth factor), insulin, insulin-like growth factors (IGF-1and/or IGF-2), ciliary neurotrophic factor (CNTF), transforming growthfactors, epidermal growth factors, activity-dependent growth factor,platelet derived growth factor, neurokine, glia-derived neurotrophicfactor (GDNF), glia-derived nexin, and cholinergic enhancing factorssuch as phosphoethanolamine and thyroid hormone T.3, and DNA or RNAvectors or plasmids that encode one or more protein neurologic agents ornerve growth promoting factors. Plasmids and vectors for delivery of acoding sequence to a mammalian tissue are known to those of skill in theart.

Metal Diseases

The method of the invention can treat or prevent diseases or disorderscaused or induced by metals, such as cancer and poisoning. Such metalscan include As, Co, Cr, Ni, Hg, Pb, Fe, Cu, V, and Cd. That is, themethod of the invention can treat or prevent poisoning by (for example)lead or mercury and also excessive iron toxicity. In one embodiment, themethod of the invention can treat or prevent CNS diseases or disorderscaused or induced by metals. In another embodiment, the method of theinvention can treat or prevent diseases or disorders not of the CNS butcaused or induced by metals, such as heart disease, blood vesseldisease, and gland disease. In another embodiment, the method of theinvention reduces poisoning of a subject by at least one metal. Inanother embodiment, the method of the invention protects a subject fromat least one carcinogenic metal. In another embodiment, the method ofthe invention reduces toxic actions of metal ions in a subject,particularly toxic actions due to Fe⁺⁺, Hg⁺⁺, Cd⁺⁺, Cu⁺⁺, As⁺⁺⁺, andPb⁺⁺ ions.

Administering Agents

Administering compounds according to the method of the invention caninclude formulating the compounds or compositions as pharmaceuticalcompositions and administering the pharmaceutical compositions to amammalian host, including a human patient, in a variety of forms adaptedto the chosen route of administration. The compounds are preferablyadministered in combination with a pharmaceutically acceptable carrier.The compounds can be administered at one of a variety of dosessufficient to provide an effective amount at the desired point of actionof the agent. Doses for humans and other mammals can range from about0.001 mg/kg to about 100 mg/kg, preferably from about 0.01 mg/kg toabout 10 mg/kg, preferably from about 0.1 mg/kg to about 1-10 mg/kg.

A related use of the methods of the invention is to protectpharmacological agents in formulation. The pharmacological agents may befor therapeutic, diagnostic, or other purposes.

The compounds can be administered by known techniques, such as orally,intranasally, parentally (including subcutaneous injection, intravenous,intramuscular, intrasternal or infusion techniques), by inhalationspray, dermally, transdermally, intrathecal, intracerebroventricular,buccal, sublingual, topically, by absorption through a mucous membraneor through the skin, or rectally, in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsor vehicles. Pharmaceutical compositions of the invention can be in theform of suspensions or tablets suitable for oral administration, nasalsprays, eye drops, nose drops, creams, sterile injectable preparations,such as sterile injectable aqueous or oleagenous suspensions orsuppositories.

Controlled or sustained release systems can also be employed. Forexample, compositions can include a polymer or other substance thatenhances controlled or sustained release. Controlled or sustainedrelease systems can include a polymer disk, such as evac disks,microspheres, and copolymers. Preferred controlled release polymers arepoly(lactide:glycolide) and poly(ethylene-co-vinyl acetate).

For oral administration as a suspension, the compositions can beprepared according to techniques well known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

In addition to the typical pharmacological methods for oraladministration, the agents employed in the methods of the invention canbe administered as a component of a nutritional or food supplement. Thenutritional or food supplement can also include any other ingredientstypical of a nutritional or food supplement, such as flavorings,stabilizers, and the like.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For intranasal administration, the compositions can be formulatedaccording to techniques well known in the art. The means of applying apharmaceutical composition intranasally can be in a variety of formssuch as a powder, spray or nose drops.

For transdermal administration, the compositions can be formulatedaccording to techniques well known in the art. Delivery of thecomposition through the skin can be accomplished by delivery means wellknown in the art, including transdermal patch, an ointment, aniontophoretic patch or device, and the like.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Preferred administration routes include orally, parenterally, as well asintravenous, intramuscular or subcutaneous routes.

Intraocular administration through the use of an ointment or eye dropsis preferred for treatment of a glandular disease or disorder of theeye, such as a lachrymal gland disease disorder like Sjogren's Syndromeor dry eye syndrome.

Solutions or suspensions of the compounds can be prepared in water,isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions or dispersions or sterile powdersincluding an active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.In all cases, the ultimate dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumincluding, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols and the like, vegetableoils, nontoxic glyceryl esters, and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption—for example, aluminummonostearate hydrogels and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsin the required amount in the appropriate solvent with various otheringredients as enumerated above and, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

Administering Agents to the Brain

Administering agents, e.g. a protective agent, a pyrophosphate analog,an agent that directly or indirectly affects a mAChR, and/or aneurologic agent, according to the method of the invention includesadministering agents to a mammalian host in a manner that allows theagents to exert their effect in the CNS. Many agents useful for themethod of the invention can be absorbed into the blood stream andreadily cross the blood brain barrier.

However, some agents useful for the method of the invention cannot pass,or have difficulty passing, the blood brain barrier. Such agents can beadministered as “prodrugs” which can cross the blood brain barrier, andupon or after entry into the CNS, the prodrug is converted to the activeagent. Agents that can cross the blood brain barrier without difficultycan also be administered as prodrugs.

To deliver the agent to the CNS, the agent alone or in combination withother substances as a pharmaceutical composition may be administered tothe spinal cord and to the cerebral vesicles according to intrathecaland intracerebrovascular administration methods known in the art. Suchpharmaceutical compositions can also be administered to the nasalcavity, under the tongue, or onto the eye. The composition may bedispensed intranasally, sublingually, or conjunctivally as a powdered orliquid nasal spray, nose drops, a gel or ointment, through a tube orcatheter, by syringe, by packtail, by pledget, or by submucosalinfusion. The agent may be combined with a polymer or other substancethat enhances controlled or sustained release of the agent. Inparticular, agents can be delivered to the brain by intranasaladministration as described in X.-Q. Chen et al. (1998) J. Alzheimer'sDisease 1:35-44 and W. H. Frey II et al. (1997) Drug Delivery 4:87-92;the disclosures of which are incorporated herein by reference.

The optimal concentration of the active agent will necessarily dependupon the specific agent used, the characteristics of the patient and thenature of the disease or condition for which the treatment is to beused.

The carrier of the composition may be any material which is otherwisepharmaceutically-acceptable and compatible with the active ingredientsof the composition. Where the carrier is a liquid, it is preferred thatthe carrier is hypotonic or isotonic with nasal, oral, or conjunctivalfluids and have a pH within the range of 4.5-7.5. Where the carrier isin powdered form, it is preferred that the carrier is also within anacceptable non-toxic pH range.

The pharmaceutical composition may be formulated as a powder, granules,solution, ointment, cream, aerosol, powder, drops, or a controlled orsustained release composition such as a polymer disk. The solution maybe sterile, isotonic or hypotonic, and otherwise suitable foradministration by injection or other means. In addition to the agent,the solution may contain appropriate adjuvants, buffers, preservativesand salts. The powder or granular forms of the pharmaceuticalcomposition may be combined with a solution and with diluting,dispersing or surface active agents. Solutions such as nose or eye dropsmay contain an antioxidant, a buffer, and the like. Further controlledrelease polymers may be used to regulate the delivery of the agent.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1 Protection of Muscarinic Acetylcholine Receptor(mAChR) in Cell Free Systems Materials and Methods

Membrane mAChR Preparation

Membranes rich in mAChRs were prepared by a modification of the methodused by Marks and Collins (Characterization of nicotine binding in mousebrain and comparison with the binding of a-bungarotoxin andquinuclindinyl benzilate, Mol. Pharmacol. 22:544-564, 1982). Gray matterfrom nondemented adult human frontal cortex was homogenized in 9 vol of50 mM Tris-HCl, pH 7.4, using 5 passes of a glass/Teflon motor-drivenhomogenizer. The homogenate was centrifuged at 27 000×g for 20 mm at 4°C., and the subsequent pellet resuspended in 9 vol of cold deionizedwater with 5 passes of the homogenizer. The resuspension was incubatedat 37° C. for 5 min, then was centrifuged as before. The resultingpellet was resuspended, incubated and centrifuged again as above. Thefinal pellet was weighed, resuspended at 15% w/v in 50 mM Tris-HClbuffer, aliquoted in small portions, flash-frozen in liquid nitrogen andstored at −70° C. for subsequent assays to determine content and bindingcapacity. Before use in binding assays, the thawed membrane preparationwas briefly rehomogenized with 10 passes in a glass/glass homogenizer. Atypical mAChR membrane preparation bound 300 μmol [³H]quinuclidinylbenzilate ([³H]QNB)/g protein.

Inhibitor Preparation

Gray matter obtained from the frontal cortex of cases with AD washomogenized in 9 vol of 1% trifluoroacetic acid (TFA) for 40 s at 4° C.in a Waring blender, then centrifuged at 1200×g for 10 min at 4° C. Theresulting supernatant fraction was centrifuged at 11 000×g for 100 minat 4° C. The 11 000×g supernatant fraction was centrifuged at 100 000×gfor 100 min at 4° C., then the 100 000×g supernatant fraction wasconcentrated using a SpeedVac and resuspended in 0.1% TFA to half theoriginal tissue volume. The 100 000×g supernatant fraction wastransferred to a Spectra/Por 3 dialysis membrane bag (3500 daltoncutoff), and dialyzed against 20 vol of 0.1% TFA at 4° C. for 24 h withgentle stirring. The resulting <3500 Da fraction (dialysate) wasconcentrated by SpeedVac to half the original tissue volume. The <3500Da fraction (endogenous inhibitor) was then frozen in liquid nitrogenand stored at −70° C. for subsequent assays to determine protein countand inhibitor activity. Protein activity was measured using thebicinchononic acid (BCA) protein assay method, essentially as describedby Smith et al. (Measurement of protein binding using bicinchonic acid,Ann. Biochem. 150: 76-85, 1985). A typical inhibitor preparationcontained about 4 mg/ml protein and approximately twice theconcentration of inhibitor found in the original tissue.

Inhibitor Activity Assay

Inhibitor activity was measured using a modification of the method ofFields et al. (Cardiac muscarinic receptors, J. Biol. Chem.253:3251-3258, 1978) to assess the binding of [³H]QNB, a mAChRantagonist, or [³H]-oxotremorine M, a mAChR agonist. In general, bindingconditions consisted of 50 mM Tris-HCl, pH 7.4 at 37° C., 10 mM reducedglutathione (GSH), 75 μ/ml membrane and 2×10⁻¹⁰ M [³H]QNB or 3 nM[³H]-oxotremorine M, with and without addition of inhibitor. To controlfor non-specific binding, 12.5 μM atropine sulfate (a mAChR antagonist)was added to several tubes. Subtracting nonspecific binding from totalbinding yielded specific binding.

Pyrophosphate, imidodiphosphate, adenylylimidodiphosphate,guanylimidodiphosphate, and tripolyphosphate were dissolved in distilledwater. Bilirubin, biliverdin, and heme were dissolved in DMSO. Carnosol,myricetin, and quercetin were dissolved in ethanol. Catalase andperoxidase were dissolved in an aqueous, preferably buffered, solution.

Enough water was added to all other reaction components in each tube tomake 4 ml total. The binding reaction was initiated by adding [³H]QNB or[³H]oxotremorine-M, mixing the tubes briefly, and then incubating thetubes at 37° C. for [³H]QNB or at room temperature for[³H]-oxotremorine-M. The reaction time for [³H]QNB was one hour in mostexperiments. In some experiments, the mAChR was preincubated with eitherthe endogenous LMW inhibitor or heme plus peroxide in the presence orabsence of the therapeutic agent being tested. The effect of thetherapeutic agent on receptor function was then assessed in a bindingassay, which for [³H]QNB was conducted at 37° C. for 40 min, and for[³H]-oxotremorine-M was conducted at room temperature for 20 min. After60 min, the binding reaction was terminated by adding 5 ml of cold 50 mMTris buffer, pH 7.4, to each tube and chilling the tubes in an ice bath.The tube contents and one 15 ml rinse of cold 50 mM Tris buffer, pH 7.4,were filtered through Whatman GF/B glass fiber filters using a Brandelharvester. The filters were placed in Optiflour scintillation flour andcounted in a Beckman LS-6500 scintillation counter set for tritiumdetection.

Results

The data resulting from the methods presented above and the resultspresented in FIGS. 1-20 are discussed in more detail below.

Pyrophosphate:

Pyrophosphate protects the mAChR from inactivation by the LMW inhibitoror by the combination of heme and peroxide. Pyrophosphate protected thereceptor from both loss of antagonist (³H-QNB) binding (FIGS. 1 and 2)and agonist (³H-Oxotremorine M) binding (FIG. 3). Approximately 1 μMpyrophosphate provides 50% protection. Pyrophosphate also protects themAChR from damage by PbCl₂. Approximately 57 μM pyrophosphate provides50% protection (FIG. 20).

Imidodiphosphates:

Imidodiphosphate (FIG. 4), guanylimidodiphosphate (FIG. 5), andadenylylimidodiphosphate (FIG. 6) all protect the mAChR frominactivation by the LMW inhibitor.

Polyphosphates:

Polyphosphates, such as tripolyphosphate (FIG. 7), protect the mAChRfrom inactivation by the LMW inhibitor.

Bisphosphonates:

Bisphosphonates, such as pamidronate (FIG. 19), protect the mAChR frominactivation by the LMW inhibitor.

Bilirubin and Biliverdin:

Bilirubin protects the mAChR from inactivation by the LMW inhibitor orby the combination of heme and peroxide. Approximately 0.7 μM bilirubinprovides 50% protection of the receptor from loss of antagonist (³H-QNB)binding (FIGS. 8 and 9) and 1.9 μM provides 50% protection from loss ofagonist (³H-Oxotremorine M) binding (FIG. 10).

biliverdin at 3 μM provides 50% protection of the mAChR (FIG. 11).

Carnosol, Quercetin, and Myricetin:

Carnosol (FIG. 12), quercetin (FIG. 13), and myricetin (FIG. 14) allprotected the mAChR from inactivation. Carnosol provided 100% protectionat 1 μM. while quercetin and myricetin provided 50% protection at 0.24μM and 0.4 μM respectively.

Catalase and Peroxidase

Catalase protected the mAChR from inactivation by the LMW inhibitor orby the combination of heme and peroxide (FIGS. 15 and 16, respectively).As little as 0.34 units/mL of catalase provided 50% protection frominactivation by heme and peroxide.

A peroxidase, specifically glutathione peroxidase, protected the mAChRfrom inactivation by the LMW inhibitor or by the combination of heme andperoxide (FIGS. 17 and 18, respectively). Glutathione peroxidase at 0.5units/mL provided 71% protection from inactivation by heme and peroxide.

Conclusion

The results indicate that pyrophosphate, imidodiphosphates,polyphosphates, bisphosphonates, bilirubin, biliverdin, carnosol,quercetin and myricetin protect a receptor and increase the ability ofagents to bind a receptor. Particularly, the results demonstrate theability of these agents to protect a muscarinic receptor from theeffects of endogenous LMW inhibitor, heme and metals and increase theability of muscarinic agonists and antagonists to bind a mAChR,suggesting that these agents can be used effectively to protect otherreceptors and increase the efficacy of other agents.

Because the mAChR is essential for memory and learning, the specificdemonstration that these agents can protect the human brain mAChR frominactivation and increase agonist binding indicates that these agentshave therapeutic potential for the treatment of cognitive and memorydisorders including those associated with aging, such as Alzheimer'sdisease.

Example 2 Protection of the mAChR in Cell Culture

Various systems for determining the protection of a mAChR in cellculture are known in the art. Such cell culture systems can be used todetermine if mAChR is protected from a damaging agent or conditionaccording to the method of the invention. For example, by administeringa mAChR antagonist or agonist alone or in combination with one or moreprotective agent and/or one or more pyrophosphate analog, one of skillin the art can determine if a mAChR is protected by the one or moreprotective agent and/or one or more pyrophosphate analog.

Example 3 Protection of the mAChR Receptor in Animals

Various systems for determining the protection of a mAChR in animals areknown in the art. Such animal systems can be used to determine if mAChRis protected according to the method of the invention. For example, byadministering a mAChR antagonist or agonist alone or in combination withone or more protective agents and/or one or more pyrophosphate analogs,one of skill in the art can determine if a mAChR is protected by the oneor more protective agents and/or one or more pyrophosphate analogs.

Example 4 Increased Efficacy of Neurologic Agents in Model Systems

Various models systems for determining the efficacy of neurologic agentsare known in the art. Such model systems can be used to determine if theefficacy of a neurologic agent is increased by the method of theinvention. For example, by administering one or more neurologic agentsalone or in combination with one or more protective agents and/or one ormore pyrophosphate analogs, one of skill in the art can determine if theefficacy of the one or more neurologic agents is increased whenadministered with one or more protective agents and/or one or morepyrophosphate analogs within the parameters of the model systemaccording to techniques known in the art.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1-80. (canceled)
 81. A method for protecting and treating at least onemuscarinic receptor from dysfunction resulting from free radical damagein a subject in need thereof, comprising: administering an effectiveamount of bisphosphonate directly to the CNS of the subject andbypassing the blood-brain barrier by intranasally administering to theupper one-third of the subject's nasal cavity the effective amount ofbisphosphonate; and protecting or treating the muscarinic receptors fromthe dysfunction resulting from free radical damage in the subjects inneed thereof.
 82. The method of claim 81, wherein the dysfunctionfurther comprises damage to the ability of the muscarinic receptors tomediate the inhibitory and excitatory effects of the neurotransmitteracetylcholine wherein the at least one dysfunctional muscarinic receptorcontributes to at least one disease or disorder.
 83. The method of claim82, further comprising protecting and treating the muscarinic receptorsthat mediate the effects of acetylcholine in the heart.
 84. The methodof claim 82, further comprising protecting and treating the muscarinicreceptors that mediate the effects of acetylcholine in smooth muscle.85. The method of claim 82, further comprising protecting and treatinginvolves the muscarinic receptors that mediate the effects ofacetylcholine in blood vessels.
 86. The method of claim 82, furthercomprising protecting and treating the muscarinic receptors that mediatethe effects of acetylcholine in glands.
 87. The method of claim 82,further comprising protecting and treating the muscarinic receptors thatmediate the effects of acetylcholine in presynaptic and postsynapticneurons.
 88. The method of claim 82, further comprising protecting andtreating the muscarinic receptors that mediate the effects ofacetylcholine in targets selected from the group consisting of theheart, smooth muscle, blood vessels, glands, and presynaptic andpostsynaptic neurons.
 89. The method of claim 82, wherein the subjectsuffers from Alzheimer's disease.
 90. The method of claim 82, whereinthe subject is protected from or treated for dysfunction of muscarinicreceptors that contribute to at least one of cancer, diseases ordisorders of the heart, smooth muscles, blood, blood vessels, glands, orbones.
 91. The method of claim 90, wherein the subject suffers from atleast one of hypertension, myocardial infarction, ischemic heartdisease, congestive heart failure, cardiac arrhythmias, irritable bowelsyndrome, diverticular disease, urinary incontinence, esophagealachalasia, chronic obstructive airways disease, xerostomia, diabetesmellitus, Sjogren's syndrome, Paget's disease, or hereditaryhematochromatisis.
 92. The method of claim 89, wherein the subject istreated to improve memory and cognition contributed to by at least onedysfunctional muscarinic receptor.
 93. The method of claim 81, furthercomprising combining the effective amount of bisphosphonate with atleast one of a bilirubin, biliverdin, carnosol, quercetin, myricetin, abioflavinoid, a combination thereof, or a pharmaceutically acceptablesalt thereof.
 94. The method of claim 81, further comprisingadministering heme oxygenase, a vector encoding a heme oxygenase, hemeoxygenase-1, a vector encoding a heme oxygenase-1, heme oxygenase-2, avector encoding a heme oxygenase-2, a biliverdin reductase, a vectorencoding a biliverdin reductase, a catalase, a vector encoding acatalase, a peroxidase, a vector encoding a peroxidase, or a combinationthereof.
 95. The method of claim 93, further comprising administering aheme binding protein.
 96. The method of claim 95, wherein the hemebinding protein comprises hemopexin, a lipoprotein, or a combinationthereof.
 97. The method of claim 81, further comprising administering aheme binding protein.
 98. The method of claim 97, wherein the hemebinding protein comprises hemopexin, a lipoprotein, or a combinationthereof.
 99. The method of claim 81, wherein the dose of bisphosphonateadministered to the subject is within the range of about 0.001 mg/kg toabout 100 mg/kg.
 100. The method of claim 81, wherein the dose ofbisphosphonate administered to the subject is within the range of about0.01 mg/kg to about 10 mg/kg.
 101. The method of claim 81, wherein thedose of bisphosphonate administered to the subject is within the rangeof about 0.1 mg/kg to about 1 mg/kg.
 102. The method of claim 82,further comprising protecting and treating the muscarinic receptors thatmediate the effects of acetylcholine in the brain.
 103. The method ofclaim 82, further comprising protecting and treating the muscarinicreceptors that mediate the effects of acetylcholine in the spinal cord.104. The method of claim 81 wherein the dysfunction is resulting fromoxidative stress and protecting or treating the muscarinic receptorsfrom oxidative stress.
 105. The method of claim 81, wherein thedysfunction is resulting from metal toxicity and protecting or treatingthe muscarinic receptors from metal toxicity.